By performing integrated studies on orf-I sequence in 160 HTLV-1A infected individuals and animal testing on the infectivity of molecular clones carrying polymorphism that result in preferential expression of p8 or p12, we established that efficient HTLV-1A persistence and spread in vivo requires the combined functions of the orf-I proteins. We also found that p8 is essential for productive infection of monocytes and that the expression of both proteins renders infected cells completely resistant to the MHC-class I restricted cytotoxic CD8 killing. Cell-associated HTLV-1 can be transmitted by at least three different mechanisms: virological synapse, cellular conduits including Tunneling Nano Tubes, and biofilm. However, the contribution of each of these mechanisms to viral transmission remains unknown. We have demonstrated that the HTLV-1 p8 increases viral transmission by increasing both cellular conduits and Tunneling Nano Tubes. More recently, we found that inhibition of p8-medaited TNT formations by the nucleoside analog cytarabine (cytosine arabinoside, AraC) decreases viral transmission by 30%, thereby providing a treatment to partly curb the spread of HTLV-1 in vivo. We have synthesized the cDNAs derived from the doubly spliced rex-orf-I mRNAs of HTLV-1C that juxtapose the first exon of Rex in frame with orf-I and demonstrated that it produces p16, a protein that increases autophagy. We constructed a chimeric virus by swapping the Cla-1-Sal-1 fragment, which contains both the entire orf-I and most of the orf-II of HTLV-1A, with that of HTLV-1C. The resultant molecular clone is a replicating virus designated as HTLV-1A/CDF. We plan to create a second HTLV-1A/C by substituting the entire 3' end (HTLV-1A/C) and generating mutants of both chimeric viruses to inhibit the splicing of the rex-orf-I mRNA (HTLV-1A/CDF?16, and HTLV-1A/CDF?16,) as a control. The viral DNA clones will be transduced in the 729 B cell line that supports HTLV-1 replication and will be used to infect primary CD4+ T cells. We plan to perform functional studies on monocytes with lentiviruses that express p8 or p16 on primary monocytes and are infected by HTLV-1A WT and the orf-I knockout mutant, the chimeric HTLV-1A/CDF WT, or rex-orf-I knock-out virus (HTLV-1A/CDF?16). We will assess inflammasome activation, autophagy, and the level and type of inflammatory cytokines and chemokines produced by primary monocytes and T cells in vitro. We have preliminary data that demonstrate that our HTLV-1A molecular clone can be transmitted to humanized NOD/SCID-yc-/- mice45 using infected, irradiated CD4+ T cells. We observed proliferation of human CD25+ CD4+ T cells engrafted in the humanized mice that causes extensive infiltration of these CD4+ T cells in vital organs such as the spleen, high viral burden, weight loss, and death. To explore HTLV-1 clonality in hu-Mice, we applied an optimized high-throughput sequencing (HTS) method to map viral integration sites in the human genome and simultaneously measure the abundance of the corresponding clones. The CD4+ T cell proliferation is polyclonal, as expected. We do not anticipate differences in the ability of HTLV-1A and HTLV-1A/C to cause this proliferative disease in mice, since the Tax is virtually identical in the two viruses. However, these studies will be prerequisite to demonstrate viral infectivity of the chimeric HTLV-1A/C in vivo before they can be used in studies in non-human primates. In addition, this small animal model may be foundational in testing the extent of inhibition of viral transmission by cytarabine in vivo. We have demonstrated that HTLV-1A WT and the HTLV-1 orf-I knockout viruses infect monocytes in vitro and macaques in vivo, but the HTLV-1 orf-I knockout does not appear to persist. We obtained PBMCs infected with HTLV-1A WT or the HTLV-1A orf-I knockout38 virus, cultured them for 3 days, and measured the ability of adherent cells (macrophages) to produce cytokines. The blood was collected at a timepoint when both animals were positive for viral DNA in PBMCs (weeks 8-10). We found higher levels of IL-1?, IL-6, and IL-8 in the animal infected with the HTLV-1A orf-I knockout than that with the HTLV-1A WT, demonstrating that the absence of orf-I results in a qualitatively different inflammatory profile in vivo, as also demonstrated in vitro in. We plan to extend this study by infecting 4 macaques with HTLV-1A and 4 with HTLV-1A orf-I knockout viruses to follow the inflammatory profiles caused by the two viruses in detail. We also plan to infect additional macaques with the HTLV-1A/CCS and the HTLV-1A/CCS?16 if warranted by the data. In all the animal studies, we will collect lung biopsies, bronchial alveolar lavage, blood, lymph nodes, gut biopsies, and spinal fluid to quantitate viral burden and differences in systemic inflammatory profiles. To compare the inflammatory profiles of ex vivo monocytes from macaques and humans infected by HTLV-1A and HTLV-1C, we have established collaborative efforts with Australian physicians at the Alice Springs Hospital that care for HTLV-1C infected Aborigines and with the IMSUT Hospital in Tokyo and Steve Jacobson at the NIH, who both care for HTLV-1A infected people, to study their inflammatory profiles directly in plasma or by short term cultures of PBMCs. Our collaboration with Australian researchers is a large pan-Australian consortium to share PBMCs, culture protocol, Luminex data, and reagents for the comparison of human and animal data.